Acylated oxyntomodulin peptide analog

ABSTRACT

A novel peptide analog of acylated oxyntomodulin and its uses are disclosed. A pharmaceutical composition containing the novel peptide analog is useful and effective for preventing and treating obesity or overweightness, or diabetes accompanied by obesity and overweightness. The peptide analog and a composition containing the peptide analog are superior to those of wild-type oxyntomodulin in dual agonism on GLP-1 and glucagon receptors and longer in vivo half-life. A pharmaceutical composition containing the peptide analog is effective in the treatment of metabolic diseases such as obesity and diabetes mellitus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 16/638,970filed Feb. 13, 2020, which is a National Stage of InternationalApplication No. PCT/KR2018/009425 filed Aug. 16, 2018, claiming prioritybased on Korean Patent Application No. 10-2017-0103798 filed Aug. 16,2017 and Korean Patent Application 10-2018-0095717 filed Aug. 16, 2018.

TECHNICAL FIELD

The present invention relates to oxyntomodulin peptide analogs andpharmaceutical composition comprising the same for the treatment orprevention of obesity, overweight, or non-insulin-dependent diabetesaccompanying said conditions.

BACKGROUND ART

Metabolic diseases, or metabolic syndrome, are usually caused byabnormalities in the metabolism of glucose, fat, proteins, and others.The term refers to a various diseases caused by abnormalities in glucoseand fat metabolism, including cancer, diabetes, bone metabolismdisorders, fatty liver, obesity, and cardiovascular disease. Accordingto the 2001 report of the National Cholesterol Education Program (NCEP)of the United States and 2012 publications of International DiabetesFederation (IDF), diagnosis of metabolic syndrome requires the presenceof 3 or more of the following 5 factors: (1) abdominal obesity indicatedby a waist circumference of 102 cm (NCEP) or 94 cm (IDF) for males and88 cm (NCEP) or 80 cm (IDF) for females; (2) hypertriglyceridemiaindicated by triglyceride level of 150 mg/dL or above; (3) HDLcholesterol level at or lower than 40 mg/dL (male) or 50 mg/dL (female);(4) hypertension indicated by a blood pressure of 130/85 mmHg or higher;(5) a fasting glucose level of 110 mg/dL or higher.

According to the World Health Organization (WHO), worldwide obesity ratehas more than doubled from 1980 to 2014; 39% of adults aged 18 years orolder (38% of male and 40% of female) were obese in 2014. Obesity andoverweight is caused by energy imbalance between caloric intake andoutput, causes of which include increased consumption of foods with highfat content and high energy density and reduced physical activity due tomodem work and lifestyle, changes in modes of transportation, andincreased urbanization. Diabetes rate has also rapidly increased; 4.7%of adults aged 18 years or older had diabetes in 1980, compared to 8.5%in 2015. Diabetes rate is increasing more rapidly in middle class andlow-income nations and is among major causes of blindness, renalfailure, cardiac arrest, and strokes.

Glucagon is a hormone produced by alpha cells of the pancreas. It worksto raise the concentration of glucose by stimulating gluconeogenesis andpromoting the breakdown of glycogen stored in liver. When liver-storedglycogen becomes depleted, glucagon stimulates liver and kidney tosynthesize new glucose. It is also known to affect appetite suppressionand breaking down of triglyceride storage into fatty acids, causingincreased metabolism, thereby affecting body weight loss(Diabetes.co.uk. the global diabetes community, Anim Sci J. 2016; 87(9):1090-1098).

Glucagon-like peptide-1 (GLP-1), a glucagon derivative, is a peptidehormone which reduces blood glucose. GLP-1 is secreted in L-cells of thesmall intestine after food intake. It has a very short half-life of nolonger than 2 minutes. It is reported that glucose increases secretionof GLP-1, which induces insulin secretion by pancreatic beta cells,ultimately controlling blood glucose level and improving beta cellfunctions. GLP-1 also suppresses secretion of glucagon, inhibits gastricemptying, and reduces food intake (Physiol Rev. 2007; 87(4):1409-1439).Novo Nordisk's liraglutide is human GLP-1 derivative which has beendeveloped to treat type 2 diabetes and obesity indications and is to beinjected once per day. Liraglutide is a long-acting GLP-1 receptoragonist which binds to the same receptors as endogenous GLP-1,stimulating insulin secretion, thereby modulating blood glucose leveland reducing appetite, thus inhibiting body weight gain and reducingtriglycerides. Liraglutide has been marketed in the U.S. and Europe asVictoza for type II diabetes and Saxenda for obesity (Expert RevCardiovasc Ther. 2015; 13(7):753-767). Exenatide, lixisenatide,albiglutide, and dulaglutide also have been developed for the treatmentof diabetes. However, such GLP-1 receptor agonists are reported to causeside effects such as nausea, vomiting, appetite reduction, headache,constipation, and abdominal bloating (Korean J Med. 2014; 87(1):9-13).

Oxyntomodulin is a peptide derived from proglucagon, a precursor ofglucagon. Oxyntomodulin consists of 37 amino acid peptides, includingthe complete 29 amino acids of glucagon, and is known to be a dualagonist that binds both to GLP-1 and glucagon receptors. It producesbody weight loss effect by reducing food intake and increasing energymetabolism. Oxyntomodulin is known to be more effective than selectiveGLP-1 receptor agonists at lowering body weight. It has been reportedthat the risk of hyperglycemia caused by a rise in glucose due toglucagon receptor activation may be offset by insulin secretion of GLP-1receptors. Oxyntomodulin has been reported to reduce food intake andbody weight, and to improve energy expenditure and glucose metabolism innon-clinical testing (Diabetes. 2009; 58(10):2258-2266). In a clinicalstudy, oxyntomodulin showed body weight loss effects of 2.3 kg onaverage when administered subcutaneously for 4 weeks, 3 times per day,to overweight and obese patients (Diabetes. 2005; 54:2390-2395). It hasbeen shown to produce significant insulin secretion and blood glucoselowering effects against placebo (Diabetes. 2013; 62(Suppl. 1):A48). Inanother clinical study, oxyntomodulin reduced energy intake without sideeffects such as vomiting or appetite stimulation from continual use ofoxyntomodulin (J Clin Endocrinol Metab. 2003; 88:4696-4701).Oxyntomodulin's effectiveness at glycemic control, lowering of foodintake, and satiety promotion have garnered interests in its potentialas a new method of obesity treatment and glycemic control (Molecularmetabolism. 2014; 3:241-251). However, because oxyntomodulin, likeGLP-1, can be cleaved by dipeptidyl peptidase-IV (DPP-IV), it isunstable in vivo and has a very short in vivo half-life (J Biol Chem.2003; 278: 22418-22423).

Therefore, studies are being conducted on DPP-IV resistant oxyntomodulinderivatives that make oxyntomodulin's pharmacological and treatmenteffects long-lasting by binding to GLP-1 and glucagon receptors inbalanced and selective ways and overcome the side effects of eachhormone peptide (Diabetes. 2009; 58(10):2258-2266). Many pharmaceuticalmanufacturers including Merck, Zealand, MedImmune, and HanmiPharmaceutical are working on development of lead compounds.

Against this background, the present inventors have worked to solve theproblem described above, culminating in the present invention, whichrelates to a pharmaceutical composition for the treatment of obesity orobesity-accompanying diabetes by developing a synthetic oxyntomodulinpeptide analog with (1) DPP-IV resistance, (2) optimized metabolicstability of acylation, and (3) improved action compared to the originaloxyntomodulin on GLP-1 and glucagon receptors.

DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The object of the present invention is to provide an oxyntomodulinpeptide analog with higher activity against GLP-1 and glucagon receptorsthan oxyntomodulin and higher in vivo half-life from improved chemicalstability by acylation (Molecular metabolism 2013; 2:468-479), andpharmaceutical compositions comprising said analog to be used intreating and preventing conditions caused by or characterized byobesity, overweight, or non-insulin-dependent diabetes.

Solution to Problem

As a solution to the above-mentioned problem, the present inventionprovides a novel peptide comprising the amino acid sequence of ChemicalFormula I below, which is an oxyntomodulin peptide analog.

<Chemical Formula I> (SEQ ID NO: 49)His-X₁-Gln-Gly-Thr-Phe-Thr-Ser-X₂-X₃-X₄-X₅-X₆-X₇-X8-X9-Arg-Arg-Ala-X₁₀-Asp-Phe-Val-Gln-Trp-Leu-X₁₁- X₁₂-X₁₃-X₁₄-X₁₅-X₁₆

In the formula above,

X₁ is Ser or Aib (aminoisobutyric acid);

X₂ is Asp or Z;

X₃ is Tyr or Z;

X₄ is Ser or Z;

X₅ is Lys or Z;

X₆ is Tyr or Z;

X₇ is Leu or Z;

X₈ is Asp or Z;

X₉ is Ser, Aib (aminoisobutyric acid) or Z;

X₁₀ is Gln or Z;

X₁₁ is Met or Leu;

X₁₂ is Asn or Arg;

X₁₃ is Thr or Ala;

X₁₄ is Lys or Z;

X₁₅ is RNRNNIA (SEQ ID NO:51) or absent;

X₁₆ is Z or absent, if X₁₅ exists;

the C-terminal amino acid may be amidated arbitrarily;

at least one or more of X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₄ and X₁₆is Z;

Z is modified form of Lys, attached at whose side chains are a polymericmoiety and spacer conjugate (“Z₁”) and a lipophilic lipid moiety (“Z₂”);where Z₁ is directly attached to Lys side chain via acyl functionalgroup; and Z₂ is attached to Lys side chain via Z₁; and Z₁ is StructuralFormula (1) or (2) below; and

Z₂ is Structural Formula (3) or (4) below.

In the present invention, the following three-letter and/orsingle-letter abbreviations are used to refer to specific amino acids:

Ala(A), Lys(K), Asn(N), Asp(D), Cys(C), His(H), Ile(I), Met(M), Ser(S),Val(V), Gly(G), Leu(L), Pro(P), Thr(T), Phe(F), Arg(R), Tyr(Y), Trp(W),Glu(E), Gln(Q), Aib(aminoisobutyric acid).

In the present invention, “oxyntomodulin” refers to the peptide madefrom pre-glucagon, the precursor to glucagon. Naturally-occurringoxyntomodulin has the following amino acid sequence:HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA (SEQ ID NO:1).

Z₁, which is one of the components of Z in the present invention, may bein the form of a copolymer having polyethylene glycol as polymericmoiety, ethylene glycol as monomer, ethanolamine, and lactic acid; or, apoly-amino acid copolymer comprising glycine and serine as monomers. Thepoly-amino acid may be of the amino acid sequence GGSGSG (SEQ ID NO:52). The chemical compound of the present invention may comprise two ormore repeating units of the above-mentioned polymeric moiety.

Furthermore, Z₁ may have a functional group at one terminal end to beattached to any residue or side chain of the above-mentionedoxyntomodulin peptide analog. Preferably, it is an acyl group, in whichcase it may be attached to the amino group of side chain via an amidebond. In some embodiment examples, Z₁ may have a functional group at oneterminus to bond to a spacer. Preferably, it is amino group, in whichcase it may form an amide bond to the spacer's carboxy group.Preferably, Z₁ is water soluble and thus may be amphiphilic orhydrophilic.

A spacer in the present invention is L-glutamic acid residue, whoseγ-carboxylic acid is covalently bonded to Z₁ (polymeric moiety) andwhose α-amino group may be covalently bonded to Z₂ (lipophilic lipidmoiety), and it may have 2 or more repeating units. The Z₂ lipid moiety,which is another component of Z, directly attaches to the spacer. Thespacer directly attaches to the polymeric moiety. The polymeric moietymay directly attach to the side chain of amino acid residue on X₂, X₃,X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₄ or X₁₆ of the oxyntomodulin peptideanalog of the present invention. Preferably, Lys may be used as theamino acid residue that enables this attachment.

Furthermore, Z₂ comprises C₁₄-C₂₀ saturated hydrocarbon chain, whoseterminal carbon is in the form of carboxylic acid, primary amide, orcarboxylic acid covalently bonded to any single amino acid. In thiscase, the hydrocarbon chain may be branched or linear. The functionalgroup needed for the hydrocarbon chain to attach to the spacer formspart of the above-mentioned lipid moiety and may comprise acyl,sulfonyl, N atom, O atom, S atom, or the likes.

Therefore, the spacer may be attached by ester, sulfonyl ester,thioester, amide or sulfonamide. Preferably, the hydrocarbon chain isattached to the amino group of the spacer in the form of an amide bondvia acyl group; therefore, the hydrocarbon chain may be part of alkanoylgroup form in particular.

While no limitation of the scope of interpretation to a particulartheory is intended, the mechanism of improved in vivo half-life of thepresent invention is believed to involve its lipophilic lipid moietybonding to albumin in the bloodstream, preventing the compound of thepresent invention from reacting as a substrate for various lyases in thebloodstream.

In the present invention, one or more amino acid side chains of theoxyntomodulin peptide analog is attached to lipophilic lipid moiety viapolymeric moiety and spacer. Such chemical modification can inducepharmaceutically beneficial effects such as increasing in vivoavailability and/or half-life and/or increasing bioavailability ofoxyntomodulin peptide analog of the present invention.

A novel peptide comprising the amino acid sequence of Chemical Formula Iabove may be prepared by taking naturally-occurring oxyntomodulin andsubstituting Ser residue at position 2 with Aib. In this example of thepresent invention, Aib is introduced to the X₁ position of theoxyntomodulin peptide analog. This compound is thought to be moreresistant to dipeptidyl peptidase IV than naturally-occurringoxyntomodulin. Ultimately, the oxyntomodulin peptide analog of thepresent invention shows improved in vivo stability compared tonaturally-occurring oxyntomodulin.

Also, above-mentioned acylated oxyntomodulin peptide analog of thepresent invention may be prepared by substituting Met residue of naturaloxyntomodulin with Leu at position 27, Asn with Arg at position 28, andThr with Ala at position 29. For example, Compound 12 (SEQ ID NO: 13) ofthe examples below is prepared by substituting X₁₁ with Leu, X₁₂ withArg, and X₁₃ with Ala.

In the present invention, preferable oxyntomodulin peptide analog is anovel peptide comprising the amino acid sequence of Chemical Formula I-1below.

<Chemical Formula I-1> (SEQ ID NO: 50)HX₁QGTFTSDX₃SKYLDX₉RRAX₁₀DFVQWLX₁₁X₁₂X₁₃X₁₄X₁₅X₁₆

In the above amino acid sequence,

X₁ is Ser or Aib (aminoisobutyric acid);

X₃ is Tyr or Z;

X₉ is Ser, Aib or Z;

X₁₀ is Gln or Z;

X₁₁ is Met or Leu;

X₁₂ is Asn or Arg;

X₁₃ is Thr or Ala;

X₁₄ is Lys or Z;

X₁₅ is RNRNNIA (SEQ ID NO: 51) or absent;

If X₁₅ exists, X₁₆ is Z or absent, and C-terminal may be amidated; and

Z is as defined in Chemical Formula I above.

Embodiment examples comprising peptide analogs of Chemical Formula Iabove include, but are not limited to, Compound 1 (SEQ ID NO: 2),Compound 2 (SEQ ID NO: 3), Compound 3 (SEQ ID NO: 4), Compound 4 (SEQ IDNO: 5), Compound 5 (SEQ ID NO: 6), Compound 6 (SEQ ID NO: 7), Compound 7(SEQ ID NO: 8), Compound 8 (SEQ ID NO: 9), Compound 9 (SEQ ID NO: 10),Compound 10 (SEQ ID NO: 11), Compound 11 (SEQ ID NO: 12), Compound 12(SEQ ID NO: 13), Compound 13 (SEQ ID NO: 14), Compound 15 (SEQ ID NO:16), Compound 16 (SEQ ID NO: 17), Compound 17 (SEQ ID NO: 18), andCompound 18 (SEQ ID NO: 19).

Also, oxyntomodulin peptide analog of the above-mentioned ChemicalFormula I according to the present invention may have within its aminoacid sequence one or more intramolecular cross-link(s); i.e., X₉ and X₁₀may form a cyclic peptide via intramolecular bonding (lactamization,di-sulfide bond) or via a cross-linker.

Therefore, another object of the present invention is to provide a noveloxyntomodulin peptide analog comprising the amino acid sequence ofChemical Formula II below.

<Chemical Formula II> (SEQ ID NO: 53)His-X₁₇-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X₁₈-Ser-Lys-Tyr-Leu-Asp-X₁₉-Arg-Arg-Ala-X₂₀-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys

In the formula above,

X₁₇ is Ser or Aib (Aminoisobutric acid);

X₁₈ is Z;

X₁₉ is Asp, Glu, Cys, Hcy (Homocysteine), Lys or Orn (Ornithine);

X₂₀ is Asp, Glu, Cys, Hcy (Homocysteine), Lys or Orn (Ornithine);

X₁₉ and X₂₀ may form a cyclic peptide via intramolecular bond orcross-linker, in which case, the cyclic peptide has either a lactam ringformed by an amide bond between two residues, a di-sulfide ring formedby a di-sulfide bond between two residues, or a cross-linked ring formedby a cross-linker bond between two residues;

C-terminal amino acid may optionally be amidated;

Z is modified form of Lys, at whose side chain polymeric moiety andspacer assembly (“Z₁”) and lipophilic lipid moiety (“Z₂”) are attached,where Z₁ is directly attached to Lys side chain via acyl functionalgroup, and Z₂ is attached to Lys side chain via Z₁, and Z₁ is StructuralFormula (1) or (2) below; and

Z₂ is Structural Formula (3) or (4) below.

The intramolecular bond between X₁₉ and X₂₀ is intramolecular lactamring formation bond if X₁₉ and X₂₀ are Asp (or Glu) and Lys (or Orn)respectively or Lys (or Orn) and Asp (or Glu) respectively, orintramolecular di-sulfide ring formation bond if X₁₉ and X₂₀ are Cys (orHcy) and Cys (or Hcy);

When X₁₉ and X₂₀ are Cys (or Hcy) and Cys (or Hcy) respectively, thecross-linker forms a ring by bonding to the thiol functional group atboth Cys (or Hcy) side chains, in which case, the cross-linker is C₁-C₆linear or branched chain alkyl, C₃-C₈ saturated or unsaturatedcycloalkyl, C₆-C₁₀ aryl, or C₅-C₁₂ heteroaryl or fused heterocyclicaryl; preferably, it is:

R is hydrogen or C₁-C₆ linear or branched alkyl chain;

When X₁₉ and X₂₀ are Asp (or Glu) and Asp (or Glu) respectively, thecross-linker forms a ring by an amide bond to the carboxyl group of bothAsp (or Glu) side chains, in which case, the cross-linker is di-aminoC₁-C₆ linear or branched chain alkyl, di-amino C₃-C₈ saturated orunsaturated cycloalkyl, aminopiperidine, piperazine, di-amino C₆-C₁₀aryl, or di-amino C₅-C₁₂ heteroaryl or fused heterocyclic aryl; andpreferably, it is:

R is hydrogen or C₁-C₆ linear or branched alkyl;

When X₁₉ and X₂₀ are Lys (or Orn) Lys (or Orn) respectively, thecross-linker forms a ring by an amide bond to the amine group at bothLys (or Orn) side chains, in which case, the cross-linker is di-carbonylC₁-C₆ linear or branched chain alkyl, di-carbonyl C₃-C₈ saturated orunsaturated cycloalkyl, di-carbonyl C₆-C₁₀ aryl or di-carbonyl C₅-C₁₂heteroaryl or fused heterocyclic aryl, and preferably, it is:

R is hydrogen or C₁-C₆ linear or branched chain alkyl;

When X₁₉ and X₂₀ are Asp (or Glu) and Lys (or Orn) respectively, or Lys(or Orn) and Asp (or Glu) respectively, the cross-linker links Asp (orGlu) by amide bond between the carboxyl group of the Asp (or Glu) sidechain and amine functional group of the cross-linker; the amine group ofthe Lys (or Orn) side chain forms an amide bond with the carboxylfunctional group of the cross-linker to form a ring; in this case, thecross-linker is alpha amino acids (such as Gly, Val, Leu, Ile), betaamino acids, carbonyl C₁-C₆ linear or branched chain alkylamine,carbonyl C₃-C₈ saturated or unsaturated alkylamine, carbonyl piperidine,aminobenzoyl, carbonyl C₆-C₁₀ arylamine or carbonyl C₅-C₁₂heteroarylamine or fused heterocyclic arylamine; and preferably, it is:

and

R is hydrogen or C₁-C₆ linear or branched chain alkyl.

Embodiment examples comprising the peptide analog of Chemical Formula IIcomprising one or more intramolecular cross-link(s) within the aminoacid sequence of Chemical Formula I above includes, but are not limitedto, Compound 14(SEQ ID NO: 15), Compound 19(SEQ ID NO: 20), Compound20(SEQ ID NO: 21), Compound 21(SEQ ID NO: 22), Compound 22(SEQ ID NO:23), Compound 23(SEQ ID NO: 24), Compound 24(SEQ ID NO: 25), Compound25(SEQ ID NO: 26), Compound 27(SEQ ID NO: 28), Compound 28(SEQ ID NO:29), Compound 29(SEQ ID NO: 30), Compound 30(SEQ ID NO: 31), Compound31(SEQ ID NO: 32), Compound 32(SEQ ID NO: 33), Compound 33 (SEQ ID NO:34), Compound 34(SEQ ID NO: 35), Compound 35(SEQ ID NO: 36), Compound36(SEQ ID NO: 37), Compound 37(SEQ ID NO: 38), and Compound 38(SEQ IDNO: 39).

The cross-linking of the peptide of the above Chemical Formula II mayform as a chemical covalent bond or an interionic interaction withinresidues in each of any two amino acids spaced 3 amino acids apart inthe sequence of Chemical Formula I or between any functional groupswithin side chain. Preferably, the cross-linking may be amino acid sidechains of X₁₉ residue and X₂₀ residue forming a lactone ring, lactamring, or di-sulfide ring; or, it may be amino acid side chains of X₁₉residue and X₂₀ residue linking to a cross-linker, forming a ring.

A novel peptide comprising the amino acid sequence of theabove-mentioned Chemical Formula II may be prepared by taking anaturally-occurring oxyntomodulin and substituting the Ser residue atposition 16 with either Cys or Hcy; substituting the Gln residue atposition 20 with Cys or Hcy; and forming inter-molecular di-sulfidering. For example, Compound 28 (SEQ ID NO: 29), one of the embodimentexamples below, is prepared by substituting X₁₉ with Cys, substitutingX₂₀ with Cys, and forming a di-sulfide ring within the molecule.

Also, the above-mentioned acylated oxyntomodulin peptide analogaccording to the present invention may be prepared by taking anaturally-occurring oxyntomodulin and substituting the Ser residue atposition 16 with Cys or Hcy, substituting Gln residue at position 20with Cys or Hcy, and linking the two residues with a cross-linker toform a ring. For example, Compound 22 aSEQ ID NO: 23), one of theembodiment examples below, is prepared by substituting X₁₉ with Cys,substituting X₂₀ with Cys, and linking the two residues with across-linker to form a ring.

Also, the above-mentioned acylated oxyntomodulin peptide analog of thepresent invention may be prepared by taking a naturally-occurringoxyntomodulin and substituting its Ser residue at position 16 with Aspor Glu, substituting the Gln residue at position 20 with Lys or Orn, andforming an intra-molecular lactam ring. For example, Compound 26 (SEQ IDNO: 27), one of the embodiment examples below, is prepared bysubstituting X₁₉ with Asp, X₂₀ with Lys, and forming an intramolecularlactam ring; and Compound 14 (SEQ ID NO:15), Compound 19 (SEQ ID NO:20),Compound 20 (SEQ ID NO:21), Compound 24 (SEQ ID NO:25), and Compound 25(SEQ ID NO:26) are prepared by substituting X₁₉ with Glu, X₂₀ with Lys,and forming an intramolecular lactam ring.

Also, the above-mentioned acylated oxyntomodulin peptide analog of thepresent invention may be prepared by taking a naturally-occurringoxyntomodulin and substituting its Ser residue at position 16 with Aspor Glu, substituting Gln residue at position 20 with Lys or Orn, andlinking the two residues with a cross-linker to form a ring. Forexample, Compound 37 (SEQ ID NO:38) and Compound 38 (SEQ ID NO:39) areprepared by substituting X₁₉ with Asp, X₂₀ with Lys, and linking the tworesidues with a cross-linker to form a ring; and Compound 33 (SEQ IDNO:34), Compound 34(SEQ ID NO:35), Compound 35 (SEQ ID NO:36), andCompound 36 (SEQ ID NO:37) are formed by substituting X₁₉ with Glu, X₂₀with Lys and linking the two residues with a cross-linker to form aring.

Also, the above-mentioned acylated oxyntomodulin peptide analog of thepresent invention may be formed by taking a naturally-occurringoxyntomodulin and substituting its Ser residue at position 16 with Lysor Orn, Gln residue at position 20 with Asp or Glu, and forming anintra-molecular lactam ring. For example, Compound 21 (SEQ ID NO: 22),one of the embodiment examples below, is prepared by substituting X₁₉with Lys, X₂₀ with Glu, and forming an intra-molecular lactam ring; andCompound 27 (SEQ ID NO:28) is prepared by substituting X₁₉ with Lys, X₂₀with Asp, and forming an intramolecular lactam ring.

Also, the above-mentioned acylated oxyntomodulin peptide analog may beprepared by taking a naturally-occurring oxyntomodulin and substitutingits Ser residue at position 16 with Lys or Orn, Gln-20 with Asp or Glu,and linking the two residues with a cross-linker to form a ring. Forexample, Compound 32 (SEQ ID NO:33), one of the embodiment examplesbelow, is prepared by substituting X₁₉ with Lys, X₂₀ with Glu, andlinking the two residues with a cross-linker to form a ring.

Also, the above-mentioned acylated oxyntomodulin peptide analog may beprepared by taking a naturally-occurring oxyntomodulin and substitutingits Ser residue at position 16 with Asp or Glu, Gln-20 with Asp or Glu,and linking the two residues with a cross-linker to form a ring. Forexample, Compound 23 (SEQ ID NO:24), Compound 29 (SEQ ID NO:30), andCompound 30(SEQ ID NO:31), a few of the embodiment examples below, areprepared by substituting X₁₉ with Glu, X₂₀ with Glu and linking the tworesidues with a cross-linker.

Also, the above-mentioned acylated oxyntomodulin peptide analog may beprepared by taking a naturally-occurring oxyntomodulin and substitutingits Ser residue at position 16 with Lys or Orn, Gln residue at position20 with Lys or Orn, and linking the two residues with a cross-linker toform a ring. For example, Compound 31 (SEQ ID NO:32), one of theembodiment examples below, is prepared by substituting X₁₉ with Lys, X₂₀with Lys and linking the two residues with a cross-linker to form aring.

It is believed that such intramolecular cross-linking stabilizes thealpha helix structure of the peptide, increasing its selectivity forGLP-1 receptor and/or glucagon receptor or increasing pharmacologicalefficacy (ACS Chem Biol. 2016; 11:324-328).

The oxyntomodulin peptide analog of the present invention may bechemically modified. In particular, each amino acid residue constitutingthe peptide may be directly connected to various spacers or linkers.Also, each residue may undergo a chemical reaction such as alkylation,disulfide bond formation, metal complexation, amidation, esterification,oxidation, and reduction to be modified to the respective chemicalproduct.

In particular, any carboxy-terminus or amino-terminus present in thestructure of the oxyntomodulin peptide analog may undergo reactions suchas esterification, amidation, and acylation to yield an analog.Moreover, oxyntomodulin peptide analog of the present invention may beprovided as an acid addition salt of any amine group in its structure ora carboxylate salt of any carboxyl group in its structure, or alkaliaddition salt thereof.

Also, the present invention relates to a pharmaceutical compositioncomprising the above-mentioned peptide analog as active ingredient andcomprising pharmaceutically acceptable excipient for the prevention andtreatment of obesity or overweight and diabetes accompanying saidconditions.

The term “prevention” in the present invention refers to any and allactions to inhibit or delay the development of the target condition ordisease. The term “treatment” in the present invention refers to any andall actions to mitigate, improve, or alleviate the symptoms of acondition or disease that has developed.

In particular, as the peptide analog of the present invention is a dualagonist of both glucagon receptors and GLP-1 receptors, it shows bothGLP-1's effects on food intake and glucagon's effects on fat metabolismand energy spending. Therefore, the pharmaceutical composition for thetreatment of obesity or overweight and diabetes accompanying saidconditions comprising the peptide analog of the present invention mayinduce medically beneficial effects on weight management by thecombination of its excessive fat removal and food intake inhibitioneffects.

Also, the peptide analog of the present invention and the pharmaceuticalcomposition comprising said peptide analog may be used to prevent ortreat diabetes accompanying obesity by lowering blood glucose. Inparticular, it may be used to treat non-insulin-dependent diabetesaccompanying obesity, or type II diabetes. While no limitation of thescope of interpretation to a particular theory is intended, thepharmaceutical composition comprising the peptide analog of the presentinvention is highly active on GLP-1 receptors which lowers bloodglucose, and thus ultimately useful for glycemic control.

Therefore, the pharmaceutical composition comprising the peptide analogof the present invention may be administered alone or in combinationwith other related pharmaceuticals for direct or indirect treatment ofany condition caused by or characterized by overweight, such astreatment and prevention of obesity, morbid obesity, pre-operativemorbid obesity, obesity-related inflammation, obesity-relatedgallbladder disease, obesity-induced sleep apnea, andobesity-accompanying diabetes. Also, the pharmaceutical compositioncomprising the peptide analog of the present invention may beadministered alone or in combination with other related pharmaceuticalsto prevent conditions that may result from the effect of weight or maybe related to such an effect, such as metabolic syndrome, hypertension,atherosclerosis-induced dyslipidemia, atherosclerosis, arteriosclerosis,coronary heart disease, or stroke.

“Administration” in the present invention refers to introduction of asubstance for treatment to a patient with a suitable method. Thepharmaceutical composition comprising the peptide analog of the presentinvention may be administered via various routes and in various formsthat enable delivery of the drug to the target tissue and the achieveintended efficacy thereof, including but not limited to, intraperitonealadministration, intravenous administration, intramuscularadministration, subcutaneous administration, Intradermal administration,oral administration, topical administration, intranasal administration,intrapulmonary administration, and intrarectal administration.

The pharmaceutical composition comprising the oxyntomodulin peptideanalog of the present invention may comprise various pharmaceuticallyacceptable excipients, including: binders, lubricants, disintegrants,excipients, solubilizers, dispersants, stabilizers, suspending agents,colorants, flavorings, and the like in the case of oral administration;a combination of buffers, preservatives, analgesics, solubilizers,isotonic agents, stabilizers, and the like in the case of injection; andbases, excipients, lubricants, preservatives, and the like in the caseof topical administration.

Examples of carriers, excipients, and diluents that may be used in theformulation of the oxyntomodulin peptide analog of the present inventioninclude: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,erythritol, maltitol, starch, acacia, alginate, gelatin, calciumphosphate, calcium silicate, cellulose, methyl cellulose,microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate ormineral oil.

The pharmaceutical composition comprising the oxyntomodulin peptide ofthe present invention may be prepared in various ways by combining withthe carriers described above. For example, it may be prepared in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, and the like for oral administration; unit dose ampoules ormulti-dose forms for injection; and solutions, tablets, pills, capsules,and sustained-release preparations.

The dosage range according to the present invention varies depending onfactors such as the patient's weight, age, sex, health, diet, excretionrate, and severity of the condition. For an adult patient, appropriatedosage may be between 0.001 to 500 mg/kg per day.

Advantageous Effects of Invention

The present invention provides a novel acylated oxyntomodulin peptideanalog; the peptide analog of the present invention is superior tonatural oxyntomodulin in activity on both GLP-1 receptors and glucagonreceptors. Particularly, the present invention shows higher biologicalactivity as a glucagon receptor agonist than as a GLP-1 receptorsagonist.

Accordingly, the pharmaceutical composition comprising the novelacylated oxyntomodulin peptide analog of the present invention may beusefully applied in the prevention or treatment of conditions caused orcharacterized primarily by obesity or overweight. Furthermore, it mayalso be used for the purpose of preventing or treatingnon-insulin-dependent obesity accompanying obesity or overweight.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the results of body weight loss efficacyevaluation in mice by single injection of acylated oxyntomodulin analogaccording to the present invention.

FIGS. 2 a and 2 b are graphs showing the results of body weight lossefficacy evaluation in mice by repeated injection of acylatedoxyntomodulin peptide analog according to the present invention for oneweek;

FIG. 2 a shows the body weight loss results;

FIG. 2 b shows the cumulative food intake results.

FIGS. 3 a and 3 b are graphs showing the results of body weight lossefficacy evaluation in rats by repeated injection of acylatedoxyntomodulin peptide analog according to the present invention for 5days;

FIG. 3 a shows body weight loss results;

FIG. 3 b shows cumulative food intake results.

FIGS. 4 a and 4 b are graphs showing the body weight loss efficacyevaluation results in mice by repeated injection of acylatedoxyntomodulin peptide analog according to the present invention for 10days;

FIG. 4 a shows body weight loss results;

FIG. 4 b shows cumulative food intake results.

FIGS. 5 a and 5 b are graphs showing the body weight loss efficacyresults in mice by repeated injection of acylated oxyntomodulin peptideanalogs according to the present invention for 10 days;

FIG. 5 a shows body weight loss results;

FIG. 5 b shows cumulative food intake results.

FIGS. 6 a and 6 b are graphs showing the body weight loss efficacyevaluation results in mice by repeated injection of the acylatedoxyntomodulin peptide analogs according to the present invention for 1week;

FIG. 6 a shows body weight loss results;

FIG. 6 b shows cumulative food intake results.

FIGS. 7 a, 7 b, 7 c and 7 d are graphs showing the body weight lossefficacy results in mice by repeated injection of acylated oxyntomodulinpeptide analogs according to the present invention for 2 weeks;

FIGS. 7 a and 7 c show body weight loss results;

FIG. 7 b shows cumulative food intake results the acylated oxyntomodulinpeptide analogs indicated in 7 a;

FIG. 7 d shows cumulative food intake results of the acylatedoxyntomodulin peptide analogs indicated in 7 c.

FIGS. 8 a and 8 b are graphs showing the body weight loss efficacyresults in mice by repeated injection of acylated oxyntomodulin peptideanalogs according to the present invention for 2 weeks;

FIG. 8 a shows body weight loss results;

FIG. 8 b shows cumulative food intake results.

FIGS. 9 a and 9 b are graphs showing the body weight loss efficacyevaluation results in mice by repeated injection of acylatedoxyntomodulin peptide analog according to the present invention for 5days;

FIG. 9 a shows body weight loss results;

FIG. 9 b shows cumulative food intake results.

FIGS. 10 a , 10 and 10 c are graphs showing the body weight lossefficacy evaluation results in mice by repeated injection of acylatedoxyntomodulin peptide analogs according to the present invention for 4weeks;

FIG. 10 a shows body weight loss results;

FIG. 10 b shows body fat mass loss results;

FIG. 10 c shows cumulative food intake results.

FIG. 11 is a graph showing the oral glucose tolerance test results inmice of acylated oxyntomodulin peptide analogs according to the presentinvention.

FIGS. 12 a and 12 b are graphs showing the results of glycemic controlefficacy in mice by repeated injection of acylated oxyntomodulin peptideanalog according to the present invention for 6 weeks;

FIG. 12 a shows non-fasting blood glucose results;

FIG. 12 b shows glycated hemoglobin elevation inhibition results.

FIGS. 13 a and 13 b are graphs showing the results of glycemic controlefficacy in mice by repeated injection of acylated oxyntomodulin peptideanalogs according to the present invention for 4 weeks;

FIG. 13 a shows non-fasting blood glucose levels over time;

FIG. 13 b shows glycated hemoglobin elevation inhibition andimprovement.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is further described in detail by reference to thefollowing examples and experimental examples. These examples areprovided for purposes of illustration only, to help a person skilled inthe art understand the invention, and should not in any way be construedas limiting the scope of the present invention.

<Example 1>Synthesis of Acylated Oxytomodulin Peptide Analog

Peptides comprising part of the amino acids of the present invention orcatalog peptide sequences may be synthesized or purchased fromcommercial peptide synthesis companies, such as American PeptideCompany, Bachem, and Anygen.

In the present invention, an auto-synthesizer model Symphony X(synthesis scale: 0.1 mmol) by Protein Technologies Inc was used tosynthesize the acylated oxyntomodulin peptide analogs. The structures ofCompound 1 (SEQ ID NO: 2) and Compound 38 (SEQ ID NO: 39), which hareacylated oxyntomodulin peptide analogs synthesized according to thepresent invention, are shown in Table 1 and Table 2. The detailedsynthesis procedures are provided below:

A mixture of Fmoc-AA-OH (1 mmol), HBTU (1 mmol), NMM(n-methylmorpholine)(2 mmol) and DMF(7 ml) is added to a resin fromwhich Fmoc has been removed and stirred at room temperature for 1 hour.Drain the reaction solution and wash twice with 7 ml ofDMF(N,N-dimethylmethanamide). Fmoc cleavage reaction is performed twicefor 5 minutes at room temperature and washed 6 times with DMF (7 ml).This process is repeated using an autosynthesizer to couple the aminoacids.

For K(Lys) side synthesis, Fmoc-K(Dde)-OH is used for coupling. For thelast H(His), Boc-His(Trt)-OH is used for coupling. Use 2% hydralazine toremove the protected Dde and then couple PEG2, rE, C18, C18 diacid, etc.For lactam ring synthesis, amino acids incorporated into Glu(Oall) andLys(Alloc) are used for coupling. After protecting group is removed,excess HBTU and DIPEA are used to perform lactam binding. The di-sulfidering is coupled using Ser amino acid incorporated into the protectinggroup. After the protecting group is removed, di-sulfide bonding isperformed. Coupling is carried out using suitable protectinggroup-incorporated amino acids at the position to which the cross-linkeris to be introduced. After the protecting group is removed, bondingbetween the cross-linker and the two amino acids is performed usingamide coupling reagent.

To 0.1 mmol of the peptide-resin obtained above, add 8 ml of Reagent K(trifluoroacetic acid, water, thioanisole, 1,2-ethandithiol (87.5, 5.0,5.0, 2.5)) solution after cooling it to 5-10° C. Then, stir at roomtemperature for 2-3 hours. After draining, wash the resin with a smallamount of TFA. Then, the filtrates are combined and added to 100 ml ofdiethyl ether to crystallized. The resulting solid is filtered to obtaincrude peptide. The crude peptide obtained is purified by preparativeHPLC to give the desired compound.

Shimadzu AXIMA Assurance MALDI-TOF was used for molecular mass analysis;α-Cyano-4-hydroxycinnamic acid (CHCA) was used as a matrix.

TABLE 1 <STRUCTURES OF ACYLATED OXYNTOMODULIN PEPTIDE ANALOGS> Com-pound Structure Com- pound 1

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Com- pound 38

TABLE 2 <SEQUENCES AND STRUCTURES OF ACYLATED OXYNTOMODULIN PEPTIDEANALOGS> SEQ ID NO. Name Sequence 1 OXM (Orig.)HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA 2 Compound 1H-HSQGTFTSDZSKYIDSRRAQDFVQWLMNTK-OH 3 Compound 2H-HAibQGTFTSDZSKYLDSRRAQPFVQWLMNTK-OH 4 Compound 3H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 5 Compound 4H-HSQGTFTSDZSKYLDSRRAQDFVQWLMNTK-NH₂ 6 Compound 5H-HSQGTFTSDZSKYLDSRRAQDFVQWLMNTKRNRNNIA-OH 7 Compound 6H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-NH₂ 8 Compound 7H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTKRNRNNIA-OH 9 Compound 8H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 10 Compound 9H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 11 Compound 10H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 12 Compound 11H-HAibQGTFTSDZSKYLDAibRRAQPFVQWLMNTK-OH 13 Compound 12H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLLRAK-OH 14 Compound 13H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 15 Compound 14H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-OH (lactam ring formed between  E and  K ) 16 Compound 15 H-HAibQGTFTSDYSKYLDZRRAQDFVQWLMNTK-OH 17Compound 16 H-HAibQGTFTSDYSKYLDAibRRAZDFVQWLMNTK-OH 18 Compound 17H-HAibQGTFTSPZSKYLDAibRRAQDFVQWLMNTK-OH 19 Compound 18H-HAibQGTFTSDZSKYLDAibRRAQPFVQWLMNTK-OH 20 Compound 19H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-NH₂ (Lactam ring formed between E  and  K ) 21 Compound 20 H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-OH(Lactam ring formed between  E  and  K } 22 Compound 21H-HAibQGTFTSPZSKYLD K RRA E DFVQWLMNTK-OH (Lactam ring formed between  K and  E ) 23 Compound 22 H-HAibQGTFTSDZSKYLD C RRA C DFVQWLMNTK-OH(A ring formed between  C  and  C  via 1,3-phenylenedimethyl cross-link)24 Compound 23 H-HAibQGTFTSDZSKYLD E RRA E PFVQWLMNTK-OH(A ring formed between  E  and  E  via 1,4-piperazinyl cross-link) 25Compound 24 H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-NH2(Lactam ring formed between  E  and  K ) 26 Compound 25H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-OH (Lactam ring formed between  E and  K ) 27 Compound 26 H-HAibQGTFTSDZSKYLD D RRA K DFVQWLMNTK-OH(Lactam ring formed between  D  and  K ) 28 Compound 27H-HAibQGTFTSDZSKYLD K RRA D DFVQWLMNTK-OH (Lactam ring formed between  K and  D ) 29 Compound 28 H-HAibQGTFTSDZSKYLD C RRA C DFVQWLMNTK-OH(Di-sulfide ring formed between  C  and  C ) 30 Compound 29H-HAibQGTFTSDZSKYLD E RRA E DFVQWLMNTK-OH (A ring formed between  E and  E  via 1,4-phenylenediamino cross-link) 31 Compound 30H-HAibQGTFTSDZSKYLD E RRA E DFVQWLMNTK-OH (A ring formed between  E and  E  via 1,2-ethylenediamino cross-link) 32 Compound 31H-HAibQGTFTSDZSKYLD K RRA K DFVQWLMNTK-OH (A ring formed between  K and  K  via 1,4-phenylenebiscarbonyl cross-link) 33 Compound 32H-HA1 bQGTFTSDZSKYLD K RRA E DFVQWLMNTK-OH (A ring formed between  K and  E  via 4-carbonylpiperidin-1-yl cross-link) 34 Compound 33H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-OH (A ring formed between  E and  K  via 1-aminocyclohexan-4-carbonyl cross-link) 35 Compound 34H-HAi bQGTFTSDZSKYLD E RRA K DFVQWLMNTK-OH (A ring formed between  E and  K  via 4-aminobenzoyl cross link) 36 Compound 35H-HAibQGTFTSDZSKYLD E RRA K DFVQWLMNTK-OH (A ring formed between  E and  K  via glycine cross- link) 37 Compound 36 H-HAibQGTFTSPZSKYLD ERRA K DFVQWLMNTK-OH (A ring formed between  E  and  K via leucine cross-link) 38 Compound 37 H-HAibQGTFTSDZSKYLD D RRA KDFVQWLMNTK-OH (A ring formed between  D  and  K  via glycine cross-link)39 Compound 38 H-HAibQGTFTSDZSKYLD D RRA K DFVQWLMNTK-OH(A ring formed between  D  and  K  via leucine cross-link) Z is amodified form of Lys; specifically, Lys with side chain bonded topolymer, spacer, or/and lipophilic lipid. Z takes on the followingspecific forms depending on the compound: For Compounds 1-7, 12, 14-16,19, 21-23, and 26-38: Lys([(2-(2-{2-aminoethoxy)ethoxy)acetoyl)₂]-[gammaglutamicacid]-[octadecancyl]) For Compound 8:Lys([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)₂)-[gammaglutamicacid][icosnoyl]) For Compound 9:Lys([(2-(2-(2-aminoethoxy}ethoxy}acetoyl)₂)-[gammaglutamicacid]₂]-(octadecanoyl]]  For Compounds 10 and 20:Lys([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)₃]-[gammaglutamicacid]-[octadecanoyl)) For Compound 11:Lys([Gly-Gly-Ser-Gly-Ser-Gly]-[gamma glutamic acid]-[octadecanoyl]) ForCompound 13: Lys([(2-(2-(2-aminoethoxy}ethoxy)acetoyl)₂]-[gammaglutamicacid]-[17-aminocarbonylheptadecanoyl]) For Compound 17:Lys([{2-(2-(2-aminoethoxy}ethoxy)acetoyl)]-[gammaglutamicacid]-[octadecanoyl]) For Compounds 18, 24, and 25:Lys([{2-(2-(2-aminoethoxy)ethoxy)acetoyl)₄]-[gammaglutamicacid]-[octadecanoyl])

<Comparative Example 1>Synthesis of Oxyntomodulin Peptide Analog

To compare with the present invention, oxyntomodulin peptide analogshaving structural similarities were synthesized by the method ofExample 1. Compound 39 below is a non-acylated oxyntomodulin peptideanalog. Compounds 40 and 41 are oxyntomodulin peptide analogs acylatedat different positions. Compounds 42 and 46 are oxyntomodulin peptideanalogs with acylation at different terminal ends. Compound 47,disclosed in Korean Patent Publication No. 2012-139579, is non-acylatedoxyntomodulin peptide analog with a ring structure. The structures ofsynthesized oxyntomodulin peptide analogs of Compounds 39 through 47above are shown in Table 3 and Table 4.

TABLE 3 <STRUCTURES OF OXYNTOMODULIN PEPTIDE ANALOGS> Com- poundStructure Com-H-H-Aib-Q-G-T-F-T-S-D-K-S-K-Y-L-D-Aib-R-R-A-Q-D-F-V-Q-W-L-M-N-T-K-OHpound 39 Com- pound 40

Com- pound 41

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Com- pound 43

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Com- pound 46

Com- pound 47

TABLE 4 <SEQUENCES AND STRUCTURES OF OXYNTOMODULIN PEPTIDE ANALOGS>SEQUENCES AND STRUCTURES OF OXYNTOMODULIN PEPTIDE ANALOGS SEQ ID NO.Name Sequence 40 Compound 39 H-HAibQGTFTSDKSKYLDAibRRAQDFVQWLMNTK-OH 41Compound 40 H-HAibQGTFTSZYSKYLDAibRRAQDFVQWLMNTK-OH 42 Compound 41H-HSQGTFTSDYSKYLDSRRAQDFVQWLMNTZ-OH 43 Compound 42H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 44 Compound 43H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 45 Compound 44H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 46 Compound 45H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 47 Compound 46H-HAibQGTFTSDZSKYLDAibRRAQDFVQWLMNTK-OH 48 Compound 47H-HAibQGTFTSDYSKYLD E KRA K EFVQWLMNTKC-OH (Lactam ring formed between E  and  K ) Z is a modified form of Lys; specifically, Lys with sidechain bonded to polymer, spacer, or/and lipophilic lipid. Z takes on thefollowing specific forms depending on the compound: For Compounds 40 and41:Lys([((2-2-(2-aminoethoxy)ethoxy)acetoyl)₂]-[(gammaglutamicacid]-[octadecanoyl])For Compound 42:Lys([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)₂]-[gammaglutamicacid]-[15-carboxypeptadecanoyl])For Compound 43:Lys(((2-(2-(2-aminoethoxy)ethoxy)acetoyl)₂]-[gammaglutamicacid]-[17-carboxyheptadecanoyl])For Compound 44:Lys([(2-(2-{2-aminoethoxy)ethoxy}acetoyl}₂]-[gammaglutamicacid]-[19-carboxynonadecanoyl])For Compound 45:Lys([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)₂]-[gammaglutamicacid]-[15-(N-(carboxymethyl)amino)carbonylpeptadecanoyl])For Compound 46:Lys([(2-(2-(2-aminoethoxy)ethoxy)acetoyl)₂]-[gammaglutamicacid]-[15-(N-(1S)-(1-carboxy-2-methylpropyl)amino)carbonylpentadecanoyl])

<Experimental Example 1>GLP-1 and Glucagon Receptors Activation Assay

Human GLP-1 or glucagon receptors were transiently overexpressed incells, so that the analog of the present invention could activate thereceptors resulting in a rise in cyclic adenosine monophosphate (cAMP),which sequentially activates cyclic adenosine monophosphate responseelements (CRE). Then, the resulting increased luciferase activity wasevaluated as a measurement of the effect on each receptor activation.

GLP-1 and glucagon were used as positive control in respectiveevaluation. Liraglutide and Semaglutide, which are GLP-1 agonistsapproved for treatment of diabetes, and MEDI0382, an oxyntomodulinpeptide analog that is in Phase II clinical trial, (Diabetes, Obesityand Metabolism 2016; 18: 1176-1190, Lancet 2018; 391: 2607-2618) as wellas Compounds 39 through 47 above were synthesized and used asComparative Examples.

Human GLP-1 or glucagon expression vector (“OriGene” hereafter) wastransiently transfected into Chinese hamster ovary cells (CHO-K1), withplasmid DNAs that can induce expression of firefly luciferase or Renillaluciferase (respectively), using Lipofectamine Plus Reagent(Invitrogen). After 3 hours of transfection, medium was exchanged toAlpha Minimal Essential Medium (α-MEM) comprising 10% fetal bovine serum(FBS). Next day, the medium was exchanged to α-MEM comprising the analogof the present invention and 0.1% bovine serum albumin (BSA). After 6hours, dual luciferase assay reagent was added in the same amount as themedium in which the cells were submerged, and firefly luciferase andRenilla luciferase activities were continuously measured. Fireflyluciferase activity values were corrected against Renilla luciferaseactivity to yield transfection efficiency.

To measure the efficacy of receptor activity, multi-concentration testwas performed on the analog of the present invention to obtain therelative activation (%) of the maximum effect of the analog on eitherGLP-1 or glucagon, and the concentration indicating 50% activation(EC₅₀) was calculated using non-linear regression analysis. Theresulting values are shown in Table 5.

TABLE 5 <ACYLATED OXYNTOMODULIN PEPTIDE ANALOGS AND THEIR ABILITY TOACTIVATE HUMAN GLP-1 AND GLUCAGON RECEPTORS> EC₅₀(pM) On GLP-1 Onglucagon Structure Compound receptors receptors Control GLP-1 A —Glucagon — A Examples Compound 1  A B Compound 2  A A Compound 3  A ACompound 4  A B Compound 5  A B Compound 6  A A Compound 7  A A Compound8  B B Compound 9  A A Compound 10 A A Compound 11 A B Compound 12 A ACompound 13 B B Compound 14 A A Compound 15 A A Compound 16 A A Compound17 A A Compound 18 A A Compound 19 A A Compound 20 A A Compound 21 A ACompound 22 A A Compound 23 A A Compound 24 A B Compound 25 A A Compound26 A A Compound 27 A A Compound 28 C C Compound 29 A A Compound 30 A ACompound 31 A A Compound 32 A A Compound 33 A A Compound 34 A A Compound35 A A Compound 36 A A Compound 37 A A Compound 38 A A ComparativeOxyntomodulin B B Examples Liraglutide B — Semaglutide B — MEDI0382 A BCompound 39 D D Compound 40 C D Compound 41 C D Compound 42 C D Compound43 D D Compound 44 C D Compound 45 C C Compound 46 C C Compound 47 A B *A: below 10 pM; B: 10-100 pM; C: 100-1000 pM; D: above 1000 pM

Experimental results show that the compounds of acylated oxyntomodulinanalogs according to the present invention have significantly lower EC₅₀values for GLP-1 and glucagon receptors than the endogenousoxyntomodulin hormone, indicating superior activity on GLP-1 andglucagon receptors. They also showed superior activity on GLP-1receptors compared to Liraglutide and Semaglutide, which are currentdiabetes drugs in the market. They also showed better activity onglucagon receptors and similar activity on GLP-1 receptors compared toMEDI0382, which is an oxyntomodulin peptide analog currently in clinicaltrial.

Compound 3, an acylated oxyntomodulin peptide analog according to thepresent invention, showed a much higher activity on GLP-1 and glucagonreceptors than comparative example Compound 39, a non-acylatedoxyntomodulin peptide analog. As such, acylation of oxyntomodulinpeptide analogs is believed to have a significant effect on activityincrease.

Also, Compound 40, a comparative example with acylation at X₂, andCompound 41, a comparative example acylated at X₁₄, showed much loweractivity on GLP-1 and glucagon receptors compared to Compounds 3, 15,and 16 according to the present invention with acylation at X₃, X₉, andX₁₀ positions, respectively. This seems to indicate that the position ofacylation of oxyntomodulin peptide analogs has a significant effect ontheir activity.

Compounds 3, 10, 17, and 18, acylated according to the presentinvention, have 1, 2, 3, and 4 (respectively)2-(2-(2-aminoethoxy)ethoxy)acetoyl groups as polymeric moiety of Z₁.There was no difference in in vitro activity based on the number of2-(2-(2-aminoethoxy)ethoxy)acetoyl groups. All showed outstandingactivity on GLP-1 and glucagon receptors.

Compounds 42, 43, and 44 are acylated comparative examples havingcarboxylic acid at the terminal of lipophilic lipid moiety as Z₂. Theyshowed lower activity on GLP-1 and glucagon receptors regardless oflipid carbon length compared to the acylated compounds according to thepresent invention such as Compound 3 whose lipophilic lipid moietyterminal is hydrocarbon. Comparative example Compounds 45 and 46 havinglipophilic lipid moiety terminal bonded to carboxylic acid and Gly andVal as Z₂ showed lower activity on GLP-1 and glucagon receptors comparedto Compound 3 and others according to the present invention whoselipophilic lipid moiety terminal is hydrocarbon, indicating thatacylated compounds with polar substituents at lipophilic lipid moietyterminal as Z₂ have low in vitro activity.

Comparative example Compound 47, a non-acylated oxyntomodulin cyclicpeptide analog, showed lower activity on glucagon receptors compared toCompound 14 according to the present invention having the same type ofintramolecular lactam ring structure. It can be inferred that acylationof oxyntomodulin peptide analogs leads to increased activity.

Compound 28, which is an acylated oxyntomodulin cyclic peptide analogaccording to the present invention, has an intramolecular disulfidebond. It showed lower activity on GLP-1 and glucagon receptors comparedto Compound 22, another acylated oxyntomodulin cyclic peptide analog. Itcan be inferred that the size of the intramolecular ring of an acylatedoxyntomodulin cyclic peptide analog affects its activity.

<Experimental Examples 2>Body Weight Loss Efficacy Evaluation by SingleInjection of Peptides According to the Present Invention

To evaluate body weight loss efficacy of the acylated oxyntomodulinpeptide analog according to the present invention, male laboratory mice(C57BL/6 mouse) were provided with diet containing high fat. Miceinduced to obesity by the high fat diet were assigned to groups by bodyweight before the experiment began. Compound 3 of the present inventionwas prepared in distilled water containing 0.01% Tween80 to a dosage of100 nmol/kg. This was injected once subcutaneously into the mouse.Afterward, body weight and food intake was measured once a day, at thesame time each day. The results are shown in FIG. 1 .

Although there was no significant difference in cumulative food intakeagainst the control group injected with vehicle only, body weight losswas seen in the group injected with Compound 3 against the controlgroup; the effect lasted for 4 days. This indicates that theoxyntomodulin peptide analog of the present invention can have a bodyweight loss effect maintained for a period of time with a singleadministration thanks to the improved chemical stability, compared tooxyntomodulin, which requires at least 1 administration per day to beeffective due to its in vivo instability and very short half-life.

<Experimental Example 3>Body Weight Loss Efficacy Evaluation by 1-WeekRepeated Injection of the Peptide of the Present

This experiment aimed to compare the body weight loss efficacy of theacylated oxyntomodulin peptide analog according to the present inventionwith commercially available diabetes/obesity treatments. Male laboratorymice (C57BL/6 mouse) were provided with diet containing high fat. Themice with high-fat-diet-induced obesity were separated into groups bybody weight before the experiment began. Compound 3, an exampleaccording to the present invention, was prepared in distilled watercontaining 0.01% Tween80 to a dosage of 100 nmol/kg or 300 nmol/kg. Ascontrol, Liraglutide, commercially available diabetes/obesity treatment,was prepared in the same vehicle to a dosage of 100 nmol/kg. Afterwards,both were injected subcutaneously for 6 days, once per day, as indicatedin Table 6. Body weight and food intake was measured once a day, at thesame time each day, to compare body weight loss efficacy of the acylatedoxyntomodulin analog against Liraglutide. On day 7, administration wasstopped and body weight recovery was checked. The results are shown inFIGS. 2 a and 2 b .

TABLE 6 Group Drug and dose administered Method of administrationComparison Liraglutide, 100 nmol/kg/QD S.C once a day × 6 groupExperimental Compound 3, 100 nmol/kg/QD group Compound 3, 300 nmol/kg/QD

There was no significant difference in cumulative food intake betweenCompound 3 and Liraglutide groups injected with identical dosage of 100nmol/kg. Nonetheless, Liraglutide showed body weight loss ofapproximately 12.2%, whereas Compound 3 showed body weight loss ofapproximately 24.6%. Also, injecting Compound 3 at 300 nmol/kg showedapproximately 37.8% of body weight loss. The acylated oxyntomodulinpeptide analog of the present invention showed more than double thedose-dependent body weight loss effect against Liraglutide andmaintained lower body weight against vehicle control group even afterdiscontinuation.

<Experimental Example 4>Body Weight Loss Efficacy Evaluation by 5-DayRepeated Injection of Peptide According to the Present Invention

This experiment aimed to find the maximum body weight loss effect of theacylated oxyntomodulin peptide analog of the present invention. Malelaboratory rats (Wistar rat) were provided with diet containing highfat. The rats with high-fat-diet-induced obesity were separated by bodyweight into groups before the experiment began. Compound 3 of thepresent invention was prepared in distilled water containing 0.01%Tween80 to a dose of 100 nmol/kg or 300 nmol/kg, which was injectedsubcutaneously once a day for a total of 4 days as indicated in Table 7.Body weight and food intake was measured once per day at the same timeeach day to measure the body weight loss efficacy over time compared tothe initial body weight. On day 5, administration was stopped and bodyweight recovery was checked. The results are shown in FIGS. 3 a and 3 b.

TABLE 7 Group Drug and dose administered Method of administrationExperimental Compound 3, 100 nmol/kg/QD S.C once a day × 4 groupsCompound 3, 300 nmol/kg/QD

The group injected with Compound 3 showed significant difference incumulative food intake against vehicle control group. Both dosagesshowed a body weight loss efficacy of approximately 12.5%. Lower bodyweight against vehicle control was maintained even afterdiscontinuation.

<Experimental Example 5>Body Weight Loss Efficacy Evaluation by 10-DayRepeatedIinjection of Peptide of the Present Invention

This experiment aimed to compare the body weight loss efficacy ofacylated oxyntomodulin peptide analog according to the present inventionagainst commercially available diabetes treatments. Male laboratory mice(C57BL/6 mouse) were given diet containing high fat. The mice withhigh-fat-diet-induced obesity were separated into groups by body weightbefore the experiment began. Compound 3 of the present invention wasprepared in distilled water containing 0.01% Tween80 to a dose of 100nmol/kg or 300 nmol/kg. Semaglutide, a commercially available diabetestreatment, was prepared in the same vehicle to a dose of 100 nmol/kg.Then, they were subcutaneously injected once every 3 days for a total of10 days as indicated in Table 8. Body weight and food intake wasmeasured once per day at the same time each day to compare the bodyweight loss efficacy of the acylated oxyntomodulin peptide analogagainst Semaglutide. The results are shown in FIGS. 4 a and 4 b .

TABLE 8 Group Drug and dose administered Method of administrationComparison Semaglutide, 100 nmol/kg/Q3D S.C once every 3 ExperimentalCompound 3, 100 nmol/kg/Q3D days × 4 Compound 3, 300 nmol/kg/Q3D

Compound 3 showed higher cumulative food intake against Semaglutide atthe same dosage (100 nmol/kg) but still showed superior body weight lossefficacy against Semaglutide (Compound 3: 13.9%, Semaglutide: 9.7%).Also, Compound 3 at 300 nmol/kg showed body weight loss of approximately16.9%. The acylated oxyntomodulin peptide analog according to thepresent invention showed superior dose-dependent body weight lossefficacy against Semaglutide.

<Experimental Example 6>Body Weight Loss Efficacy Evaluation by 10-DayRepeated Injection of Peptide According to Present Invention

This experiment aimed to compare the body weight loss efficacy ofacylated oxyntomodulin peptide analogs according to the presentinvention with varying structures—the number of polymers and presence ofcyclic peptide formation. Male laboratory mice (C57BL/6 mouse) weregiven diet containing high fat. The mice with high-fat-diet-inducedobesity were separated into groups by body weight before the experimentbegan. Compounds 3, 10, and 14 were prepared in distilled watercontaining 0.01% Tween80 to a dose of 100 nmol/kg, and then injectedsubcutaneously once every 3 days for a total of 10 days as indicated inTable 9. Body weight and food intake was measured once per day at thesame time each day to compare Compound 3 against Compounds 10 and 14 onbody weight loss efficacy. The results are shown in FIGS. 5 a and 5 b .

TABLE 9 Group Drug and dose administered Method of administrationExperimental Compound 3, 100 nmol/kg/Q3D S.C once every 3 Compound 10,100 nmol/kg/Q3D days × 4 Compound 14, 100 nmol/kg/Q3D

At same dosage, Compounds 10 and 14 showed cumulative food intakereduced by 20% and 27% respectively against vehicle control group.Cumulative food intake on Compound 3 decreased by approximately 17%. Allthree compounds showed outstanding body weight loss efficacy ofapproximately 19-22% from initial body weight. The cyclic peptideCompound 14 had slightly better results than Compounds 3 and 10.

<Experimental Example 7>Body Weight Loss Efficacy Evaluation by 1-WeekRepeated Injection of Peptide of Present Invention

This experiment aimed to compare the body weight loss efficacy ofacylated oxyntomodulin peptide analogs of varying structures—the numberof lipophilic lipid moiety or polymers within the peptide. Malelaboratory mice (C57BL/6 mouse) were given diet containing high fat. Themice with high-fat-diet-induced obesity were separated into groups bybody weight before the experiment began.

Compounds 3, 13 and 18 according to the present invention were preparedin distilled water containing 0.01% Tween80 to a dosage of 100 nmol/kgand as indicated in Table 10 injected subcutaneously once every 3 daysfor a total of 7 days. Body weight and food intake was measured once perday at the same time each day. Body weight loss efficacy of Compound 3was compared against Compounds 13 and 18. The results are shown in FIGS.6 a and 6 b .

TABLE 10 Group Drug and dose administered Method of administrationExperimental Compound 3, 100 nmol/kg/Q3D S.C once every 3 Compound 13,100 nmol/kg/Q3D days × 3 Compound 18, 100 nmol/kg/Q3D

At same dosage, Compound 18 showed similarly outstanding body weightloss efficacy to Compound 3, whereas Compound 13 did not have anysignificant effect on body weight. Compound 13 showed about 10 timesless in vitro efficacy than Compound 3 and was consistent in animaltesting, confirming the loss of body weight loss efficacy as a result ofthe terminal structure of lipophilic lipid moiety, confirming structuralimportance.

<Experimental Example 8>Body Weight Loss Efficacy Evaluation by 2-WeekRepeated Injection of Peptide According to Present Invention

This experiment aimed to compare the body weight loss efficacy ofacylated oxyntomodulin peptide analogs according to the presentinvention with varying cyclic peptide structures. Male laboratory mice(C57BL/6 mouse) were given diet containing high fat. The mice withhigh-fat-diet-induced obesity were separated into groups by body weightbefore beginning the experiment. Compounds 3, 21-25 according to thepresent invention were prepared in distilled water containing 0.01%Tween80 to a dosage of 100 nmol/kg and as indicated in Table 11subcutaneously injected once every 3 days for a total of 2 weeks. Bodyweight and food intake was measured once per day at the same time eachday. Body weight loss efficacy of Compound 3 and Compounds 21-25 wascompared. The results are shown in FIGS. 7 a through 7 d .

TABLE 11 Group Drug and dose administered Method of administrationExperimental Compound 3, 100 nmol/kg/Q3D S.C once every 3 Compound 21,100 nmol/kg/Q3D days × 5 Compound 22, 100 nmol/kg/Q3D Compound 23, 100nmol/kg/Q3D Compound 24, 100 nmol/kg/Q3D Compound 25, 100 nmol/kg/Q3D

At same dosage, Compounds 23 and 25 had higher cumulative food intakecompared to Compound 3. Compounds 21 and 22 showed similar cumulativefood intake as Compound 3. Compound 3 showed similar or better bodyweight loss compared to Compounds 21, 23 and 25. Compound 25 wasconfirmed to have better body weight loss efficacy than Compound 3. Onthe other hand, Compound 24, which showed somewhat weak action onglucagon receptor compared to other compounds in in vitro assay, showedsignificant body weight loss efficacy, at about 13.4% against initialbody weight, showing inferior body weight loss efficacy compared toCompound 3. Compound 25 showed similar level of body weight lossefficacy to Compound 3 but had lower cumulative food intake. On theother hand, Compound 22 showed similar body weight loss efficacy toCompound 3 and similar cumulative food intake. Acylated oxyntomodulincyclic peptide analogs showed different body weight loss efficacypatterns depending on their structures.

<Experimental Example 9>Body Weight Loss Efficacy Evaluation By 2-WeekRepeated Injection of Peptide of Present Invention

Continuing from Experimental Example 8, this experiment was conducted tocompare acylated oxyntomodulin peptide analogs of the present inventionin low doses. Male laboratory mice (C57BL/6 mouse) were given dietcontaining high fat. The mice with high-fat-diet-induced obesity wereseparated into groups by body weight before beginning the experiment.Compounds 3 and 22 according to the present invention were prepared indistilled water containing 0.01% Tween80 to a dosage of 10 nmol/kg or 30nmol/kg and as indicated in Table 12 injected subcutaneously once every3 days for a total of 2 weeks. Body weight and food intake was measuredonce per day, at the same time each day. Body weight loss efficacy ofCompound 3 and Compound 22 was compared. The results are shown in FIGS.8 a and 8 b .

TABLE 12 Group Drug and dose administered Method of administrationExperimental Compound 3, 10 nmol/kg/Q3D S.C once every 3 Compound 3, 30nmol/kg/Q3D days × 5 Compound 22, 10 nmol/kg/Q3D Compound 22, 30nmol/kg/Q3D

At the same dosage, Compound 22 showed higher cumulative food intakecompared to Compound 3. Compound 22 at 10 nmol/kg showed similar levelof cumulative food intake as vehicle control group but body weight lossefficacy of 7.5%. Furthermore, Compound 22 at 30 nmol/kg had higher foodintake than Compound 3 at the same dose but had similar levels of bodyweight loss efficacy. It can be inferred that Compound 22 is affectedmore by the efficacy resulting from glucagon receptor activationcompared to Compound 3.

<Experimental Example 10>Body Weight Loss Efficacy Evaluation by 5-DayRepeated Injection of Peptide of Present Invention

This experiment was conducted to compare the body weight loss efficacyof acylated oxyntomodulin peptide analog of the present invention withcommercially available diabetes/obesity treatment and an oxyntomodulinpeptide analog lead compound currently in development. Male laboratorymice (C57BL/6 mouse) were given diet containing high fat. The mice withhigh-fat-diet-induced obesity were grouped by body weight beforebeginning the experiment. Compound 3 of the present invention wasprepared in distilled water containing 0.01% Tween80 to dose of 30nmol/kg. As comparative examples, Liraglutide (commercially availablediabetes/obesity treatment) and MEDI0382 (in clinical trial) wereprepared in the same vehicle to a dose of 30 nmol/kg. Then, they weresubcutaneously injected once per day for 4 days total, as indicated inTable 13. Body weight and food intake was measured once per day, at thesame time each day. Body weight loss efficacy of the analog was comparedwith Liraglutide and MEDI0382. The results are shown in FIGS. 9 a and 9b .

TABLE 13 Group Drug and dose administered Method of administrationComparison Liraglutide, 30 nmol/kg/QD S.C once a day × 4 MEDI0382, 30nmol/kg/QD Experimental Compound 3, 30 nmol/kg/QD

Among the three substances at same dose, the Compound 3 group showedmost body weight loss from baseline (Compound 3: 18.6%, Liraglutide:12.7%, MEDI0382: 8.0%).

<Experimental Example 11>Body Weight Loss Efficacy Evaluation by 4-WeekRepeated Injection of Peptide According to Present Invention

This experiment was conducted to compare the body weight loss efficacyof acylated oxyntomodulin peptide analogs according to the presentinvention with commercially available diabetes treatment. Male obese anddiabetic laboratory mice (FATZO mouse) were given diet containing highfat, and were grouped by body weight, body fat, non-fasting bloodglucose, and glycated hemoglobin (HbA1c) before experiment. Compounds 3and 22 according to the present invention were prepared in distilledwater containing 0.01% Tween80 to a dose of 30 nmol/kg or 100 nmol/kg.Semaglutide, commercially available diabetes treatment, was prepared inthe same vehicle to a dose of 100 nmol/kg. Then, they were givensubcutaneously once every 3 days for a total of 4 weeks, as indicated inTable 14. Body weight and food intake was measured once per day, at thesame time each day. Body fat was measured at 4 weeks before autopsy tocompare the body weight and body fat reduction efficacy of oxyntomodulinpeptide analogs of the present invention against Semaglutide. Theresults are shown in FIGS. 10 a through 10 c .

TABLE 14 Group Drug and dose administered Method of administrationComparison Semaglutide, 100 nmol/kg/Q3D S.C once every 3 ExperimentalCompound 3, 30 nmol/kg/Q3D days × 10 Compound 3, 100 nmol/kg/Q3DCompound 22, 30 nmol/kg/Q3D Compound 22, 100 nmol/kg/Q3D

At both same and lower dosage (100 and 30 nmol/kg respectively),Compounds 3 and 22 of the present invention shows highly superior bodyweight reduction efficacy against Semaglutide despite higher cumulativefood intake. This is inferred to be a result of the mechanism of actionof oxyntomodulin peptide analog according to the present invention beinga dual agonist of GLP-1 and glucagon receptors, whereas Semaglutide is aGLP-1 receptor agonist.

<Experimental Example 12>Oral Glucose Tolerance Test in Mice of PeptideAccording to Present Invention

In this experiment, glucose tolerance improvement effect in malelaboratory mice (C57BL/6 mouse) of acylated oxyntomodulin peptideanalogs according to the present invention was evaluated as improvementof postprandial glycemic control. Laboratory mice were fasted the daybefore the experiment. Then, Compound 3 or 22 or 25 according to thepresent invention was prepared in distilled water containing 0.01%Tween80 and injected subcutaneously 30 minutes before glucose loading.Glucose solution was orally administered 30 minutes after the injectionof oxyntomodulin peptide analog. Whole blood glucose was measured viatail vein immediately before administering the drug and glucose, and for2 hours after glucose loading at designated times. From the results, thearea under the curve (AUC) of the blood glucose curve over time wasproduced to calculate the ratio of blood glucose AUC of the analog andcomparison against glucose control as percentages to evaluate theefficacy of glucose tolerance improvement. Experiments were conductedseparately for each compound. Compound 3 at 30 nmol/kg was used ascomparison for Compound 22 or 25. The combined results are shown in FIG.11 .

The peptide analogs showed significant, dose-dependent reduction ofblood glucose AUC at and above 30 nmol/kg. In particular, Compound 25showed significant glucose tolerance improvement efficacy and threetimes superior glucose tolerance improvement efficacy compared toCompounds 3 and 22 in all dose groups evaluated.

<Experimental Example 13>Glycemic Control Efficacy Evaluation by 6-WeekRepeated Injection of Peptide according to Present Invention

The present invention was conducted to compare the glycemic controlefficacy of acylated oxyntomodulin peptide analogs according to thepresent invention with commercially available diabetes treatment. Malelaboratory diabetes mouse models (db/db mouse) were grouped bynon-fasting glucose level, glycated hemoglobin (HbA1c), and body weightbefore experiment. Compound 3 according to the present invention wasprepared in distilled water containing 0.01% Tween80 to a dose of 100nmol/kg. Commercially available Semaglutide was prepared in same vehicleto a dose of 100 nmol/kg. Afterwards, they were injected subcutaneouslyonce every 3 days for a total of 6 weeks as indicated in Table 15.Non-fasting glucose, body weight, and food intake were measured once aweek, 24 hours after drug administration. Glycated hemoglobin wasmeasured at 3, 5 and 6 weeks. The glycemic control efficacy ofoxyntomodulin peptide analog according to the present invention wascompared with that of Semaglutide. The results are shown in FIGS. 12 aand 12 b .

TABLE 15 Group Drug and dose administered Method of administrationComparison Semaglutide, 100 nmol/kg/Q3D S.C once every 3 ExperimentalCompound 3, 100 nmol/kg/Q3D days × 16

Initially, Compound 3 of the present invention showed similar or lowerefficacy compared to Semaglutide at same dose (100 nmol/kg). However,after long-term administration, Compound 3 showed better glycemiccontrol efficacy than Semaglutide. The vehicle control group had a finalnon-fasting blood glucose of 581 mg/dL, showing very severely diabeticcondition. On the other hand, Semaglutide showed a final non-fastingglucose of 342 mg/dL; Compound 3 had a final non-fasting blood glucoseof 274 mg/dL, showing significant inhibition of glucose elevation. Finalglycated hemoglobin (HbA1c) of the vehicle control group was 5.15%points higher than initial level. In comparison, Semaglutide HbA1cincreased by 2.65% points, and Compound 3 HbA1c only 1.73% points,confirming efficacy in glycated hemoglobin elevation inhibition.Therefore, the acylated oxyntomodulin peptide analog according to thepresent invention showed superior efficacy to Semaglutide in delayingthe onset of diabetes.

<Experimental Example 14>Glycemic Control Efficacy Evaluation by 4-WeekRepeated Injection of Peptide According to Present Invention

This is the same experiment as Experimental Example 11. It was conductedto compare glycemic control efficacy of acylated oxyntomodulin peptideanalog according to the present invention in comparison to commerciallyavailable diabetes treatment. Male obese and diabetic laboratory mice(FATZO mouse) were given diet containing high fat and were grouped bybody weight, body fat, non-fasting glucose, and glycated hemoglobin(HbA1c) before experiment. Compounds 3 and 22 according to the presentinvention were prepared in distilled water containing 0.01% Tween80 to adosage of 30 nmol/kg or 100 nmol/kg. Commercially available Semaglutidewas prepared in same vehicle to a dose of 100 nmol/kg. As indicated inTable 16, both were injected subcutaneously once every 3 days for atotal of 4 weeks. Body weight and food intake was measured once per dayat the same time each day. Non-fasting glucose was measuredapproximately every 10 days, 24 after administration. Glycatedhemoglobin was measured before autopsy at 4 weeks. Glycemic controlefficacy of oxyntomodulin peptide analog of the present invention wascompared with Semaglutide. The results are shown in FIGS. 13 a and 13 b.

TABLE 16 Group Drug and dose administered Method of administrationComparison Semaglutide, 100 nmol/kg/Q3D S.C once every 3 ExperimentalCompound 3, 30 nmol/kg/Q3D days × 10 Compound 3, 100 nmol/kg/Q3DCompound 22, 30 nmol/kg/Q3D Compound 22, 100 nmol/kg/Q3D

The groups administered with Compounds 3 and 22 of the present inventionshowed outstanding glycemic control efficacy and glycated hemoglobinelevation inhibition at similar levels as Semaglutide. In addition tothe body weight loss effect shown in Experimental Example 11, theoxyntomodulin peptide analog of the present invention is shown to alsohave glycemic control effect.

The invention claimed is:
 1. An oxyntomodulin peptide analog comprisingan intramolecular cross-link at X₁₉ and X₂₀ of the amino acid sequenceof the following Formula II:His-X₁₇-Gln-Gly-Thr-Phe-Thr-Ser-Asp-X₁₈-Ser-Lys-Tyr-Leu-Asp-X₁₉-Arg-Arg-Ala-X₂₀-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Lys(SEQ ID NO: 53), wherein: X₁₇ is Aib (Aminoisobutric acid); X₁₈ is Z;X₁₉ is Asp, Glu, Cys, Hcy (Homocysteine), Lys or Orn (Ornithine); X₂₀ isAsp, Glu, Cys, Hcy (Homocysteine), Lys or Orn (Ornithine); wherein theintramolecular cross-link at X₁₉ and X₂₀ forms a cyclic peptide viaintramolecular bond or via a cross-linker, said cyclic peptide having alactam ring formed by amide bond between two residues at X₁₉ and X₂₀ ora cross-linked ring formed by cross-linker bond between two residues atX₁₉ and X₂₀; C-terminal amino acid may optionally be amidated; and Z ismodified Lys to which Z₁-Z₂ of the following formulas is attached; Z₁ isFormula (1); and

Z₂ is Formula (3);

wherein * of Formula (1) is directly bound to a side chain of Lys, andwherein ** of Formula (1) and ** of formula (3) are a bond between Z₁and Z₂.
 2. The oxyntomodulin peptide analog of claim 1, wherein theintramolecular bond between X₁₉ and X₂₀ is an intramolecular lactam ringwhen X₁₉ and X₂₀ are Asp or Glu and Lys or Orn respectively or Lys orOrn and Asp or Glu respectively.
 3. The oxyntomodulin peptide analog ofclaim 2, wherein X₁₉ and X₂₀ are Cys or Hcy and Cys or Hcy respectively,and a cross-linker bonds to the thiol functional group at both Cys orHcy side chains to form a ring; and said cross-linker is C₁-C₆ linear orbranched chain alkyl, C₃-C₈ saturated or unsaturated cycloalkyl, C₆-C₁₀aryl, C₅-C₁₂ heteroaryl, or C₅-C₁₂ fused heterocyclic aryl.
 4. Theoxyntomodulin peptide analog of claim 3, wherein the cross-linker isselected from the group consisting of

wherein R is hydrogen or C₁-C₆ linear or branched alkyl chain.
 5. Theoxyntomodulin peptide analog of claim 1, wherein X₁₉ and X₂₀ are Asp orGlu and Asp or Glu respectively, and a cross-linker forms an amide bondto the carboxylic group at both Asp or Glu side chains to form a ring;and said cross-linker is di-amino C₁-C₆ linear or branched alkyl chain,di-amino C₃-C₈ saturated or unsaturated cycloalkyl, aminopiperidine,piperazine, di-amino C₆-C₁₀ aryl, di-amino C₅-C₁₂ heteroaryl, ordi-amino C₅-C₁₂ fused heterocyclic aryl.
 6. The oxyntomodulin peptideanalog of claim 5, wherein the cross-linker is selected from the groupconsisting of

wherein R is hydrogen or C₁-C₆ linear or branched alkyl chain.
 7. Theoxyntomodulin peptide analog of claim 1, wherein X₁₉ and X₂₀ are eachLys or Orn and Lys or Orn respectively; a cross-linker forms an amidebond with the amine group at both Lys or Orn side chains to form a ring;and said cross-linker is di-carbonyl C₁-C₆ linear or branched alkylchain, di-carbonyl C₃-C₈ saturated or unsaturated cycloalkyl,di-carbonyl C₆-C₁₀ aryl, di-carbonyl C₅-C₁₂ heteroaryl, or di-carbonylC₅-C₁₂ fused heterocyclic aryl.
 8. The oxyntomodulin peptide analog ofclaim 7, wherein the cross-linker is selected from the group consistingof

and R is hydrogen or C₁-C₆ linear or branched alkyl chain.
 9. Theoxyntomodulin peptide analog of claim 1, wherein X₁₉ and X₂₀ are Asp orGlu and Lys or Orn respectively, or are Lys or Orn and Asp or Glurespectively; and a cross-linker forms an amide bond between thecarboxyl group of Asp or Glu side chain and the amine functional groupof the cross-linker; and/or the amine group at Lys or Orn side chain isconnected via an amide bond to the carboxyl functional group of thecross-linker to form a ring; and said cross-linker either an alpha aminoacid selected from the group consisting of Gly, Val, Leu, Ile; a betaamino acid; carbonyl C₁-C₆ linear or branched alkylamine; carbonyl C₃-C₈saturated or unsaturated alkylamine; carbonyl piperidine; aminobenzoyl;carbonyl C₆-C₁₀ arylamine, carbonyl C₅-C₁₂ heteroarylamine, or carbonylC₅-C₁₂ fused heterocyclic arylamine.
 10. An oxyntomodulin peptide analogof claim 9, wherein the cross-linker is selected from the groupconsisting of

and R is hydrogen or C₁-C₆ linear or branched alkyl chain.
 11. Theoxyntomodulin peptide analog of claim 1, wherein said peptide analog isCompound 14 (SEQ ID NO: 15), Compound 19 (SEQ ID NO: 20), Compound 20(SEQ ID NO: 21), Compound 21 (SEQ ID NO: 22), Compound 22 (SEQ ID NO:23), Compound 23 (SEQ ID NO: 24), Compound 27 (SEQ ID NO: 28), Compound29 (SEQ ID NO: 30), Compound 30 (SEQ ID NO: 31), Compound 31 (SEQ ID NO:32), Compound 32 (SEQ ID NO: 33), Compound 33 (SEQ ID NO: 34), Compound34 (SEQ ID NO: 35), Compound 35 (SEQ ID NO: 36), Compound 36 (SEQ ID NO:37), Compound 37 (SEQ ID NO: 38), or Compound 38 (SEQ ID NO: 39).
 12. Apharmaceutical composition comprising the peptide analog of claim 1 anda pharmaceutically acceptable excipient.
 13. A method of treating asubject with obesity or overweight comprising administering an effectiveamount of the pharmaceutical composition of claim 12 to the subject. 14.The method of claim 13, wherein the subject suffers fromnon-insulin-dependent diabetes.